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2013/10/17What to Expect TodayFlowJo :Flow Data Analysis 均泰生物科技高芳儀 Fay Kaotechsupport@gtbiotech.com.twDevelop basic knowledge of flow data &data displayFlowJoGating strategyCompensationDemo: Gate/ Report/ CompensationFrom Cells to DataFrom Cells to DataInformations collectedLight Scatter parameters-Forward Scatter: proportional to cell sizeSide Scatter: proportional to cell granularity or cellinternal complexityFluorescence parametersTime parametershttp://flow.csc.mrc.ac.uk/?page id 51
2013/10/17Flow Cytometry Standard--.FCS DataAll those information about the cells orparticles will be collected and write into a listmode file.The principal goal of the Standard is toprovide a clearly defined and uniform fileformat allowing files created by one type ofacquisition instrument to be analyzed byanother type on another computerFlowJo Features2
2013/10/17Task BarRibbon BarFluorescence Data DisplayHistogram(1D)Bivariate plots(2D)GroupsMultidimensional plots(3D)SamplesHistograms- Histograms display thedistributions of the events for oneparameter-The X--fluorescence intensity-The Y--number of cells perBivariate Displays-Dot PlotsBivariate plots display data with any two parameters,Scatter parameter or Fluorescence parameterAdjunct HistogramchannelHistogram overlaysControlEach dot corresponds one or more cell events-Pro: show rare events very well-Cons: Saturate in areas of high cell density, noindication of the relative density of the eventsA perfect way to show both histogramand the dot plot. Showing thehistogram of each parameter alongwith traditional dot plots, provide moreinfo to your data3
2013/10/17Bivariate DisplaysBivariate Displays-More Options- Show OutliersBiexponential Transformation3D- to Better Visualize Data After CompensationY XY log(x)y arcsinh(x)-Transformation is a bi-exponentialdisplay tool by introducing linear scalesinto your axes to better visualize yourdata- It does not change your Herzenbergdata!et. al. Nature Immunology 20064
2013/10/17Gating: Define Population(s) ofInterest(POI)Gating basicGating-Gate on population of interest and gate out population notof interest-Gate out debris, dead cells,doublets/cell aggregates,Focus on POI-Is both objective and subjectiveGating tools-Hierarchical gatingGating StrategyGate out DebrisDebris(NOT)Gate out doubletsSinglets2nFlowJo hierarchicalgating treePlot on FSC&SSCPOIgrandparentParentChild4n2n 2nGate out deadcellsLive cellsHAW5
2013/10/17Hierarchical GatingPowerful Analysis ToolsMonocytes120T cellsLymphocytes906030CD4 CD8 CD8 0Data Report in FlowJoVarious forms of data reportStatistic Report:excel,HTML, SQLGraphic Report: jpg,gif,png,pdf,web,movie, et. al.StatisticsBatch Analysis-Batch Gate & Statistics-Batch Report:Table Editor & Layout EditorTemplateTemplatesAnalysis of repetitiveexperimentmade easy Design aworkspace Analyze once Save as a template All gates, stats,tables and layoutsare saved for futureuse!6
2013/10/17Special Analysis PlatformsDNA Cell CycleProliferationKinetics (Calciuminflux)CompensationAll in one program, no need to install extra modulesCompensation Controls-To CorrectSpectra SpilloverWays to CompensateUncompensatedhardware compensation Out: old school Subjective: Inaccuratemanual adjustmentTrue PE total FL2 -FITC spill over Complicated Acquisition Permanent: the matrixwrote into the FCS fileand not removable withFCS 2.0Software compensation In: new generation Objective: Accuratecalculate mathematically Simplifies Acquisition Non-permanent: createand modify matrix witheaseSlide credit to Dr. Holden Maecker StanfordUniversity7
2013/10/17 30 day free trial Video Tutorial WebEx Tech BlogSoftware Compensation Set up single stainingFor analog instrument, do not use hardwarecompensation or under compensate your dataif you have toFor digital instrument, export data as FCS3.0 flowjo.typepad.comwww.flowjo.com Tech support mailboxflowjo@treestar.com均泰生物科技: techsupport@gtbiotech.com.tw8
compensation or under compensate your data if you have to For digital instrument, export data as FCS3.0 Software Compensation 30 day free trial Video Tutorial WebEx Tech Blog flowjo.typepad.com Tech support mailbox www.flowjo.com flowjo@treestar.com 均泰生物科技 : techsupport@gtbiotech.com.tw
